PROJECT SUMMARY Septins are a family of conserved GTP-binding proteins that assemble into hetero-oligomeric complexes and polymerize into filaments and other supramolecular arrangements. Septin structures associate with the plasma membrane by interacting with a specific phosphoinositide, and are involved in cell compartmentation, in cell division, and other membrane remodeling events. In budding yeast, septins establish a diffusion barrier at the neck between a mother and daughter cell, promote membrane curvature, and act as a scaffold to recruit other proteins to the site of cytokinesis. In humans, septin structures serve very similar functions; they are localized at the cleavage furrow, at the root of the primary cilium, at the base of every dendritic spine, and within the annulus in spermatozoa. Marked alterations of septin gene expression are found in solid tumors of certain tissues and septin gene translocations in mixed lineage leukemias. SEPT9 mutations are one apparent cause of hereditary neuralgic amyotrophy. Substitution mutations preventing GTP binding to SEPT12 disrupt the sperm annulus and cause male infertiity. In-depth structural and functional analysis of septins is necessary to understand the molecular mechanisms governing septin organization and function and obtain new insights to illuminate their pathophysiology. This project aims to apply novel tools and the experimental advantages of yeast to interrogate septin-based structures and eludicate fundamental properties of their organization, regulation and function that should be applicable to the highly homologous septin-based structures in human cells. Our specific aims include experimental tests of the following hypotheses. (1) Post-translational modifications (PTMs) of septins and septin-associated proteins drive the dramatic changes in septin structural organization that occur during passage through the cell division cycle. Therefore, comprehensive analysis by mass spectrometry of PTMs (especially phosphorylation and SUMOylation) that septins undergo during cell cycle progression, and subsequent genetic analysis of the physiological importance of those modifications by site-directed mutagenesis in vivo and a FRET-based method in vitro, will be conducted. (2) Sequential recruitment of septin-binding proteins exert and order the spatio-temporal changes in septin organization and function that impose proper cell morphology. Hence, comprehensive analysis of the in vivo septin interactome using a tripartite split-GFP method will be carried out. In addition, a novel clonable tag for in vivo labeling and protein localization at the ultrastructural level, which will be applied to dynamic analysis of the septin interactome using correlated light (fluorescence) and electron microscopy (CLEM), will be developed; and, (3) Association with PtdIns4,5P2 is obligatory for septin recruitment to the plasma membrane. Thus, to gain unprecedented insight as to how septins associate with a PtdIns4,5P2-containing lipid surface and how specific septin-binding proteins influence that interaction, the newest instrumentation for cryo-EM will be used to visualize these structures at near-atomic resolution.